Biocompatible phase invertable proteinaceous compositions and methods for making and using the same

ABSTRACT

Biocompatible phase invertible proteinaceous compositions and methods for making and using the same are provided. The subject phase invertible compositions are prepared by combining a proteinaceous substrate and a crosslinking agent, such as a stabilized aldehyde crosslinking agent. Also provided are kits for use in preparing the subject compositions. The subject compositions, kits and systems find use in a variety of different applications.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application of U.S.application Ser. No. 12/510,890 filed on Jul. 28, 2009; whichapplication is a continuation-in-part application of U.S. applicationSer. No. 11/877,396 filed on Oct. 23, 2007; which application is acontinuation application of U.S. application Ser. No. 10/635,847 filedAug. 5, 2003 and now issued as U.S. Pat. No. 7,303,757; whichapplication is a continuation-in-part application of U.S. applicationSer. No. 10/243,482 filed on Sep. 13, 2002 and also claims priorityPursuant to 35 U.S.C. §119 (e) to the filing date of U.S. ProvisionalApplication Ser. No. 60/401,282, filed on Aug. 6, 2002; the disclosuresof which applications are herein incorporated by reference.

INTRODUCTION

1. Field of the Invention

The field of this invention is biocompatible compositions, includingbiocompatible sealant compositions.

2. Background of the Invention

Recently, a number of sealant compositions have become available tocontrol fluid leakage at a surgical site, as well as for otherapplications. However, currently available sealant compositions maysuffer from serious limitations with regards to the field in which theycan be used, as well as their biocompatibility and their physicalproperties. Side effects, such as inflammation, acute fibrous formationat the wound site, toxicity, inability to be used in a bloody field,poor physical properties of the sealant, and poor adhesion to thesurgical site, may have a serious impact on the patient and resultantlymay play a significant role in the long term efficacy of the repair.Further, useful sealants have properties that can render them moreeffective for surgical application. Characteristics, such as the abilityto be localized to a specific location, adequately long or shortpolymerization times, and adequate in vivo resorption characteristics,are vital to a successful completion of the sealing procedure.

As such, there is a continued need for the development of newbiocompatible compositions for use as sealants, as well as for use inother applications.

SUMMARY OF THE INVENTION

Biocompatible phase invertible proteinaceous compositions and methodsfor making and using the same are provided. The subject phase invertiblecompositions are prepared by combining a proteinaceous substrate and acrosslinker. The proteinaceous substrate includes one or more proteinsand, in some embodiments, an adhesion modifier, and may also include oneor more of: a plasticizer, a carbohydrate, or other modification agent.In certain embodiments, the crosslinker is a heat-treated dialdehyde,e.g., heat-treated glutaraldehyde. Also provided are kits for use inpreparing the subject compositions. The subject compositions, kits andsystems find use in a variety of different applications.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A to 1E illustrate the in situ production of a biocomposite stentaccording to a representative embodiment of the subject invention.

FIG. 2 provides a representation of an alternative embodiment of adelivery device according to the subject invention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Biocompatible phase invertible proteinaceous compositions and methodsfor making and using the same are provided. The subject phase invertiblecompositions are prepared by combining a proteinaceous substrate and acrosslinker. The proteinaceous substrate includes one or more proteinsand, in some embodiments, an adhesion modifier, and may also include oneor more of: a plasticizer, a carbohydrate, or other modification agent.In certain embodiments, the crosslinker is a heat-treated dialdehyde,e.g., heat-treated glutaraldehyde. Also provided are kits for use inpreparing the subject compositions. The subject compositions, kits andsystems find use in a variety of different applications.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the subject components ofthe invention that are described in the publications, which componentsmight be used in connection with the presently described invention.

In further describing various aspects of the invention, the subjectphase invertible compositions are described first in greater detail,followed by a review of representative applications in which thecompositions find use, as well as a review of kits and systems that finduse in making or using the subject phase invertible compositions.

Biocompatible Phase Invertible Proteinaceous Composition

As summarized above, the subject invention provides a biocompatiblephase invertible proteinaceous composition that, over time, undergoes aphase inversion from a first, fluid state to a second, solid state. Thesubject phase invertible compositions are characterized by being capableof bonding tissue in both wet (e.g., blood) and dry environments, whereadhesion of the composition to the tissue is exceptionally strong. Afurther feature of the subject compositions is that they are welltolerated and do not elicit a substantial inflammatory response, if anyinflammatory response.

The subject phase invertible proteinaceous compositions are prepared bycombining or mixing a proteinacous substrate with a crosslinker, e.g., astabilized aldehyde crosslinker, such as a stabilized glutaraldehydecrosslinker (such as described in greater detail below). Each of theseprecursor components or compositions is now reviewed separately ingreater detail.

Proteinaceous Substrate

The proteinaceous substrate from which the subject phase invertiblecompositions are prepared is generally a fluid composition, e.g., anaqueous composition, that is made up of at least a proteinaceouscomponent and, in some embodiments, an adhesion modifier, where thesubstrate may include one or more additional components, including, butnot limited to: a plasticizer; a carbohydrate; and the like.

Proteinaceous Component

The proteinaceous component of the substrate is made up of one or moredistinct proteins. The proteins of this component may be eithersynthetic or naturally occurring proteins, where the proteins may beobtained/prepared using any convenient protocol, e.g., purification fromnaturally occurring sources, recombinant production, syntheticproduction, and the like, where in certain embodiments the proteins areobtained from naturally occurring, e.g., bovine or human, sources.Specific proteins of interest include, but are not limited to: albumins,collagens, elastins, fibrins, and the like.

The amount of protein in the substrate composition may vary, where thespecific selection of concentration is dependent on the desiredapplication and product parameters desired therefore, such as tenacity,hardness, elasticity, resorption characteristics and plateletaggregation effects. In certain embodiments, the total protein totalconcentration in the substrate compositions ranges from about 1 to 75%(w/w), such as 1-50% (w/w), including 5 to 40% (w/w).

In certain embodiments, the primary protein of the substrate compositionof this embodiment is albumin, where the albumin may be a naturallyoccurring albumin, e.g., human albumin, bovine albumin, etc., or avariant thereof. As is known in the art, the albumin may be purchased inpowdered form and then solubilized into an aqueous suspension, oralternately, may be purchased in aqueous form. Purified albumin mayderived from any one of a number of different sources including, bovine,ovine, equine, human, or avian in accordance to well known methods(ref.: Cohn et. Al, J. Amer. Chem. Soc. 69:1753) or may be purchased inpurified form from a supplier, such as Aldrich Chemical (St. Louis,Mo.), in lyophilized or aqueous form. The albumin may be derivatized toact as a carrier for drugs, such as heparin sulfate, growth factors,antibiotics, or may be modified in an effort to moderate viscosity, orhydrophilicity. Derivitization using acylating agents, such as, but notlimited to, succinic anhydride, and lauryl chlorides, are useful for theproduction of binding sites for the addition of useful molecules. Inthese embodiments where the proteinaceous component includes albumin,the albumin may be present in concentrations ranging from about 10 toabout 50% (w/w), such as from about 30 to about 40% (w/w).

In certain embodiments, the proteinaceous component also includes acollagen, e.g., a naturally occurring collagen (human, bovine) orsynthetic variant thereof. In accordance with the invention, thecollagen may be in dry or aqueous forms when mixed with the albumin.Collagen may be derivatized to increase it utility. Acylating agents,such as anhydrides or acid chlorides, have been found to produce usefulsites for binding of molecules such as growth factors, and antibiotics.When present, the collagen sometimes ranges from about 1 to about 20%(w/w), including from about 1 to about 10% (w/w), such as from about 1to about 4% (w/w), including from about 2 to 4% (w/w).

The subject proteinaceous component, as described above, may or may notinclude one or more active agents, e.g., drugs, present in it, asdesired. When present, the agent(s) may be bound to the polymers, asdesired.

Adhesion Modifier

Also present in some embodiments of the substrate is one or moreadhesion modifiers or tacking agents. Adhesion modifiers (also referredto herein as tacking agents) improve the adhesiveness of the sealant tothe biological surface. In some embodiments, the adhesion modifiers arepolymeric compounds having charged functionalities, e.g., amines, etc.Whereas numerous adhesion modifiers may be used, one of particularapplicability is polyethyleneimine (PEI). PEI is a long chain branched,alkyl polymer containing primary, secondary and tertiary amines. Thepresence of these highly ionic groups results in significant attachmentthrough ionic interactions with the underlying surface. In addition, thepresence of PEI in the substrate significantly enhances the presence ofamine terminals suitable to produce crosslinks with the crosslinkingagent. Additional adhesion modifiers of interest include, but are notlimited to: gelatin, carboxymethylcellulose, butylhydroxytoluene, etc.

In certain embodiments of the invention, adhesion modifiers are used tomodify adhesion to the biological substrate while simultaneouslycreating a procoagulant. In certain embodiments, the adhesion modifiersare present in concentrations of from about 0.1 to about 10% (w/w), suchas from about 0.5 to about 4% (w/w).

Optional Components

The above described substrate component of the subject compositions may,in certain embodiments, include one or more optional components thatmodify the properties of the phase invertible composition produced fromthe substrate and crosslinker. Representative optional components ofinterest are now discussed in greater detail below.

Plasticizing Agents

In accordance with the invention, a plasticizing agent may be present inthe substrate. The plasticizing agent provides a number of functions,including wetting of a surface, or alternately, increasing the elasticmodulus of the material, or further still, aiding in the mixing andapplication of the material. Numerous plasticizing agents exist,including fatty acids, e.g., oleic acid, palmitic acid, etc.,dioctylphtalate, phospholipids, and phosphatidic acid. Becauseplasticizers are typically water insoluble organic substances and arenot readily miscible with water, it is sometimes advantageous to modifytheir miscibility with water, by pre-mixing the appropriate plasticizerwith an alcohol to reduce the surface tension associated with thesolution. To this end, any alcohol may be used. In one representativeembodiment of this invention, oleic acid is mixed with ethanol to form a50% (w/w) solution and this solution then is used to plasticize theproteinaceous substrate during the formulation process. Whereas the typeand concentration of the plasticizing agent is dependent upon theapplication, in certain embodiments the final concentration of theplasticizing agent is from about 0.01 to 10% (w/w), including from about2 to about 4% (w/w). Other plasticizing agents of interest include, butare not limited to: polyethylene glycol, glycerine, butylhydroxytoluene,etc.

Carbohydrate Procoagulant

In certain embodiments, the substrates include a carbohydrateprocoagulant. Chitosan and derivates of chitosan are potent coagulatorsof blood and, therefore, are beneficial in formulating sealant materialscapable of sealing vascular injuries. While virtually all chitinmaterials have been demonstrated to have some procoagulant activity, inaccordance to the invention, the use of acetylated chitin is preferableas an additive for the formulation of sealant intended for bloodcontrol. Acetylation of the molecule can be achieved in a number ofdifferent ways, but one common method is the treatment ofchitosan/acetic acid mixtures with acid anhydrides, such as succinic.This reaction is readily carried out at room temperature. In accordanceto the invention, gels created in this manner combined withproteinaceous substrates and crosslinked in situ are beneficial for thecreation of a biocomposite structural member. In accordance with theteachings of this invention the carbohydrate component, e.g., chitosan,may be present in concentrations ranging from about 0 to about 20%, suchas from about 2 to about 5% (w/w).

Fillers

Fillers of interest include both reinforcing and non-reinforcingfillers. Reinforcing fillers may be included, such as chopped fibroussilk, polyester, PTFE, NYLON, carbon fibers, polypropylene,polyurethane, glass, etc. Fibers can be modified, e.g., as describedabove for the other components, as desired, e.g., to increasewettability, mixability, etc. Reinforcing fillers may be present fromabout 0 to 40%, such as from about 10 to about 30%. Non-reinforcingfillers may also be included, e.g., clay, mica, hydroxyapatite, calciumsulfate, bone chips, etc. Where desired, these fillers may also bemodified, e.g., as described above. Non-reinforcing fillers may bepresent from about 0 to 40%, such as from about 10 to about 30%.

Biologically Active Agents

Biologically active agents may be included, e.g., bone growth factors,tissue activators, cartilage growth activators, small molecule activeagents, etc.

Foaming Agent

In certain embodiments, the substrate may include a foaming agent which,upon combination with the crosslinker composition, results in a foamingcomposition, e.g., a compositions that includes gaseous airbubblesinterspersed about. Any convenient foaming agent may be present, wherethe foaming agent may be an agent that, upon contact with thecrosslinking composition, produces a gas that provides bubble generationand, hence, the desired foaming characteristics of the composition. Forexample, a salt such as sodium bicarbonate in an amount ranging fromabout 2 to about 5% w/w may be present in the substrate. Uponcombination of the substrate with an acidic crosslinker composition,e.g., having a pH of about 5, a foaming composition is produced.

Additional Modifiers

Additional modifiers may also be present. For example, blends of one ormore polymers (e.g., polyblends), such as Teflon, PET, NYLON, hydrogels,polypropylene, etc., may be present. The polyblends may be modified,e.g., as described above, to provide for desired properties. Theseadditional modifiers may be present in amounts ranging from about 0 to50%, including from about 10 to about 30%.

Crosslinker Composition and Preparation Thereof

As indicated above, the phase invertible composition is produced bycombining a proteinaceous substrate, as described above, with acrosslinker composition, where the crosslinker composition stabilizesthe proteinaceous substrate, e.g., by forming covalent bonds betweenfunctionalities present on different polypeptide strands of theproteinaceous substrate. Crosslinking typically renders the molecules ofthe composition less susceptible to chemical degradation, and as suchmodifies the resorption characteristics of the composition as well asthe biological responses induced by the presence of the composition.Numerous crosslinking agents have been identified. Representativeexamples of crosslinkers of interest include, but are not limited to:photo-oxidative molecules; carbodimides; carbonyl containing compounds,e.g., mono- and dicarbonyls, including carboxilic acids, e.g.,dicarboxylic acids, such as adipic acid, glutaric acid and the like, andaldehydes, including mono-and dialdehydes, e.g., glutaraldehyde; etc. Incertain embodiments, the crosslinker employed is an aldehydecrosslinker.

In certain embodiments, the aldehyde crosslinker is pretreated toproduce a stabilized aldehyde crosslinker, e.g., a stabilizedglutaraldehyde crosslinker. To produce a stabilized aldehydecrosslinker, such as a stabilized glutaraldehyde crosslinker, an amountof an initial aldehyde is first mixed with water at a particular pH toproduce an aqueous aldehyde composition, where the concentration ofaldehyde in this composition may range from about 1 to about 20% (w/w),including from about 7 to about 12% (w/w), and the pH may range fromabout 5 to about 10, including from about 6 to about 8, e.g., about 7.In producing a stabilized crosslinker composition, the above initialaqueous aldehyde composition is then heated to a temperature for aperiod of time sufficient to produce the desired stabilized aldehydecrosslinker composition. In certain embodiments, the composition may bemaintained at a temperature of from about 35 to about 60° C., such asfrom about 45 to about 55° C., for a period of time ranging from about 1to about 20 days, e.g., from about 1 to about 14 days, including fromabout 72 to about 120 hours. This step may be accomplished via anyconvenient protocol, e.g., by heating the initial aqueous compositionunder a nitrogen atmosphere. The product crosslinker is present in astabilized form and is therefore a stabilized aldehyde crosslinkercomposition.

Following heating, the resultant stabilized aldehyde crosslinkercomposition is cooled to room temperature and then used as a crosslinkerfor the proteinaceous substrate. An aspect of the stabilized aldehydecrosslinker composition is that no additional reducing agents arerequired to stabilize the crosslinked product upon use, since thestabilized aldehyde crosslinker composition is electrovalently in astable form.

As indicated above, an example of a stabilized aldehyde crosslinker ofthe invention is a stabilized glutaraldehyde crosslinker. In thisexample, heat treatment of glutaraldehyde, e.g., as described above,prior to use for crosslinking produces a product that is more stablepost crosslinking, eliminating the need for reduction. According to anembodiment of the invention, a stabilized aldehyde composition usefulfor crosslinking is produced by heating a solution of glutaraldehyde fora period of time. In certain embodiments of the invention, a 1-20%aqueous solution of glutaraldehyde pH 7.0 is prepared according tostandard methodology. In some instances, the solution is in the range of7-12%. An aliquot of solution is placed into an air-tight flask under anitrogen head and heated in an oven at a temperature of 35-60° C., suchas 45-55° C., for a period of 1-14 days.

Glutaraldehyde treated in this manner will form a crosslinkingcomposition that includes one or more distinct pyridinium precursorcrosslinking molecules. Pyridinium precursor crosslinking molecules thatmay be present in the crosslinking composition include those describedby the following formula:

where R₁ and R₂ are H or hydrocarbon moieties (which may includecarbonyl moieties) which are polymerization products that may varydepending on the number of distinct glutaraldehyde monomeric units thatpolymerize as a result of sequential aldol reaction and in someinstances dehydration of the glurataldehyde monomer units.

In some instances, the crosslinking molecules present in thecrosslinking composition may be defined by the formula:

wherein R₂ is a polymerization product produced by sequential aldolreaction and dehydration of one or more glutaraldehyde molecules, suchas 3 to 10 glutaraldehyde molecules, including 3 to 5 glutaraldehydemolecules.

Specific pyridinium precursor complexes of interest include:

The following reaction scheme illustrates the formation of pyridiniumprecursor complexes which may ultimately be used to produce a stablecrosslink without need for the use of any additional reducing agents.

Reaction of the stabilized glutaraldehyde crosslinking compositionproduced as described above with reactive side groups (e.g., reactiveamino groups found on lysine, arginine and/or histidine residues) ofproteins in the proteinaceous substrate crosslinks the proteins via acrosslinking structure that includes a pyridinium moiety as shown in thefollowing reaction scheme:

The above reaction scheme shows the ring structures formed uponcompletion of the pyridinium crosslinks.

Benefits of using the subject stabilized aldehyde crosslinkingcompositions include the feature that crosslinks produced using suchcrosslinking compositions are covalently bonded structures and are notsusceptible to reversal. Thus, proteins crosslinked using suchcrosslinking compositions are more stable and do not exhibit the intenseinflammatory responses noted as a result of the reversal of crosslinkswhen using non-heat treated dialdehydes.

Buffer

Upon mixture of the proteinaceous substrate and crosslinker to producethe subject phase invertible composition, buffering of the phaseinvertible composition is important for a number of reasons, e.g., tooptimize the bonding strength of the composition to the attachingsurface, to optimize the conditions necessary for internal crosslinkingto occur, etc. For example, optimum crosslinking for proteins usingglutaraldehyde crosslinkers occurs at pH range from about 6 to about 8.Buffers capable of maintaining this range are useful in this invention,as long as they do not interfere with the carbonyl terminal of thecrosslinker or modify the amine terminus of the amino acids. Forexample, phosphate buffers have a pKa value in the range of pH 7.0 anddo not interfere with the crosslinking process because they do notcontain carboxylic or amine functionalities. Phosphate buffer up to 1Min strength is suitable for use as a buffer in the present invention,where in certain embodiments the phosphate buffer is about 0.2M instrength. While phosphate buffering of the solutions is ideal for thestability of the protein substrate in applications where increasedadhesion is required, an acidic buffer may be used as well. Citratebuffers 0.1-1M and having a pH range of about 4.5 to about 6.5 have beenfound to be useful for this invention.

The buffer may be present in either the initial crosslinker component orthe initial proteinaceous substrate component, or present in bothcomponents, as desired.

Combination of Substrate and Crosslinker to Produce Phase InvertibleComposition

As summarized above, the subject phase invertible compositions areprepared by combining a proteinaceous substrate and crosslinker inappropriate amounts and under conditions sufficient for the phaseinvertible composition to be produced. The, substrate and crosslinkerare typically combined in a ratio (v/v) ranging from about 1/5 to about5/1; so that a resultant phase invertible composition is produced inwhich the total protein concentration typically ranges from about 10 toabout 60%, such as from about 20 to about 50%, including from about 30to about 40% and the total crosslinker compositions typically rangesfrom about 0.1 to about 20%, such as from about 0.5 to about 15%,including from about 1 to about 10%.

Combination of the substrate and crosslinker typically occurs undermixing conditions, such that the two components are thoroughly combinedor mixed with each other. Combination or mixing may be carried out usingany convenient protocol, e.g., by manually combining two components, byemploying a device that combines the two components, etc. Combination ormixing is typically carried out at a temperature ranging from about 20to about 40° C., such as room temperature.

Combination of the proteinaceous substrate and crosslinker as describedabove results in the production of a phase invertible composition. Byphase invertible composition is meant a composition that goes from afirst fluid state to a second non-fluid, e.g., gel or solid, state. Inthe second non-fluid state, the composition is substantially, if notcompletely, incapable of fluid flow. The phase invertible compositiontypically remains in a fluid state, following combination of thesubstrate and crosslinker components, for a period of time ranging fromabout 10 seconds to about 10 minutes, such as from about 20 seconds toabout 5 minutes, including from about 30 seconds to about 120 second,when maintained at a temperature ranging from about 15° C. to about 40°C., such as from about 20° C. to about 30° C.

Methods

The subject biocompatible phase invertible compositions are typicallyemployed in methods where a quantity of the phase invertible compositionis delivered to a particular site or location of a subject, patient orhost in need thereof. The subject, patient or host is typically a“mammal” or “mammalian,” where these terms are used broadly to describeorganisms which are within the class mammalian, including, but notlimited to, the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), ungulates (e.g., cows, pigs, sheep andhorses), lagomorpha (e.g. rabbits) and primates (e.g., humans,chimpanzees, and monkeys). In many embodiments, the animals or hosts,i.e., subjects (also referred to herein as patients) will be humans.

The quantity that is delivered to the subject in any given applicationwill necessarily vary depending on the nature of the application and useof the composition, but in certain representative embodiments rangesfrom about 1 to about 100 ml, such as from about 1 to about 50 ml,including from about 1 to about 25 ml, e.g., from about 1 to about 5 ml,e.g., such as about 3 ml.

While necessarily dependent on the particular application in which thesubject composition is being employed, the subject composition is, incertain embodiments, locally delivered to a particular region, site orlocation of the host, where the site or location may, of course, vary.Representative sites or locations include, but are not limited to:vessels, organs, and the like, including various tissue types, such asbut no limited to connective tissue, e.g., ligaments, tendons, bone,etc. Depending on the particular application, the composition may bedelivered to the site of interest manually or with a delivery device,e.g., the delivery device employed to deliver the composition instenting applications, described in greater detail below.

Utility

The subject biocompatible phase invertible compositions find use in avariety of different applications. Representative applications of thesubject phase invertible compositions include those described in U.S.Pat. Nos. 3,438,374; 5,092,841; 5,292,362; 5,385,606; 5,583,114;5,843,156; 6,162,241; 6,290,729; 6,302,898; 6,310,036; 6,329,337;6,371,975; 6,372,229; 6,423,333; 6,458,147; 6,475,182; and 6,547,806; aswell as U.S. Application Nos. 2002/0015724; 2002/0022588; 2002/0133193;2002/0173770; 2002/0183244; 2002/019490; 2002/0032143; the disclosuresof which are herein incorporated by reference.

Vascular Stenting Applications

In one application of particular interest, the subject inventionprovides methods and devices for producing a biocomposite structuralmember, e.g., a stent, in situ at a vascular site. In these embodiments,the first step is to position or place a distal end of a fluidcomposition delivery device at the vascular site where the biocompositestructure member is to be produced. The vascular site in which thestructural member is produced in the subject methods is typically adefined location or region of an arterial vessel. By arterial vessel ismeant a vessel of a vascularized animal in which blood flows away fromthe heart. In many embodiments, the arterial vessel is a cardiovascularvessel. In a specific embodiment of interest, the cardiovascular vesselis a coronary artery in which blood flows back into the heart to supplythe heart muscle.

In certain embodiments, the fluid composition delivery device is adevice that includes at its distal end first and second occlusionmembers flanking an expandable mandrel. As such, the devices of theseembodiments include, at their distal ends, first and second occlusionmembers separated by an expandable mandrel.

The first and second occlusion members may be any convenient type ofocclusion member. In certain embodiments, the occlusion members aredeployable balloons, where a variety of balloon occlusion members areknown in the relevant art and may be employed in the subject devices. Inyet other embodiments, the occlusion members are non-balloon occlusionmembers, such as occlusion members that, upon deployment, produce acollar configuration that results in blockage of fluid flow in thevessel at the location of deployment. The above described occlusionmembers are merely representative of the types of occlusion members thatmay be employed, where the only requirement is that the member serve toocclude the vessel at the region of deployment, i.e., that the membersubstantially, if not completely, stop the flow of blood into and out ofthe region that is occluded.

Positioned between the first and second occlusion members is anexpandable mandrel. As such, an expandable structure around which aphase invertible fluid may be placed and allowed to set, as describedbelow, is present between the first and second occlusion members. Theexpandable mandrel, in many embodiments, includes one or more fluidintroduction and removal ports, where these ports are holes or analogousstructures that serve as entry or exit paths for fluid to enter or leavefluid conveyance structures, e.g., lumens, that lead from the distal endof the device to a different location of the device, e.g.; to theproximal end of the device, for example a fluid reservoir in fluidcommunication with the proximal end of the device.

A feature of certain (though not all) embodiments is that the expandablemandrel expands or deploys as a function of either deployment of thefirst and second occlusion members or initiation of delivery of a phaseinvertible fluid composition to the vascular site of interest. As such,in certain embodiments the device is one in which deployment of thefirst and second occlusion members results in deployment of theexpandable mandrel. In yet other embodiments, deployment of theexpandable mandrel occurs as a function of introduction of the phaseinvertible fluid composition to the vascular site, e.g., uponintroduction of fluid into a delivery lumen of the device.

Following placement or positioning of the distal end of the device, asdescribed above, at the vascular site, the first and second occlusionmembers and expandable mandrel are deployed to produce a mold space forthe structural member to be formed in situ at the vascular site. Theproduced mold space is bounded at either end by the first and secondocclusion members. The lumen of the vessel in which the vascular site islocated serves as the outer wall of the mold space and the expandablemandrel serves as the inner wall of the mold space. As such, the moldspace defines a tubular volume of space bounded in the inner surface bythe mandrel, on the outer surface by the lumen of vessel at the vascularsite, and at the top and bottom by the first and second occlusionmembers.

In the subject methods, a phase invertible fluid composition is thenintroduced into the mold space, as defined above. While in the broadestsense, the phase invertible fluid composition may be any fluid that iscapable of phase inverting from a first fluid composition to a secondsolid composition over a period of time into a physiologicallyacceptable biocomposite structural member, e.g., a stent, in manyembodiments the phase invertible material employed is the phaseinvertible material of the present invention, as described above. Inthose embodiments where the phase invertible fluid composition is onethat is prepared from a substrate and a crosslinker, e.g., such as therepresentative composition described above, the device employed in thesubject methods may have elements for mixing or combining the substrateand crosslinker at the vascular site (for example at the point where thefluid exits a port in the mandrel), or at a position upstream of thevascular site, e.g., at a location at the proximal end of the fluiddelivery device.

Following introduction of the phase invertible material, the phaseinvertible fluid composition now present in the mold space is allowed toundergo a phase inversion to said second solid state. Next, theexpandable mandrel and occlusion members are retracted or collapsed, andthe distal end of the device is removed from the vascular site, leavingthe resultant biocomposite structural member at the vascular site. Assuch, practice of the subject methods results in the in situ productionof a biocomposite structural member at a vascular site.

Specific Representative Embodiments

FIGS. 1A to 1E provide an illustration of the practice of arepresentative method according to the subject method where abiocomposite stent is produced in situ at a vascular site having astenotic lesion on a luminal surface of an arterial vessel.

FIG. 1A provides a cross-sectional view of a coronary artery 10 showingvessel walls 12 and stenotic lesion 14 present on the luminal surfacethereof.

FIG. 1B shows placement of device 20 at the vascular site occupied bythe lesion 14, where the lesion has been compacted against the luminalsurface of the vessel, e.g., using standard balloon angioplastytechniques. Device 20 is a catheter device having a proximal occlusionballoon 22 and a distal occlusion balloon 24 flanking, i.e., separatedby, an expandable mandrel 26. Adjacent the distal occlusion balloon 24is marker band 28. Also shown is guidewire 21. Guidewire 21 and markerband 28 to aid in placement of the distal end of the device at thevascular site. Balloon lumens 23 are also shown, as is a fluid deliverylumen 25. Present on the surface of expandable mandrel 26 are aplurality of fluid entry and exit ports 27, which are used to introducefluid into and/or remove fluid from a mold space produced upondeployment of the occlusion members and expandable mandrel.

In FIG. 1C, the proximal and distal balloons and the expandable mandrelhave been deployed to produce a stent mold space 30 at the vascularsite, where the stent mold 30 is a tubular volume that is bounded ateither end by the distal and proximal occlusion balloons, on the outersurface by the lumen having the compressed lesion thereon, and on theinner surface by the expandable mandrel.

FIG. 1D shows introduction of phase invertible material 40 into the moldspace 30, e.g., via ports 27.

The introduced phase invertible fluid composition is then allowed to setor harden, following which device 20 is removed from the vascular site,leaving behind a biocomposite stent 50 depicted in FIG. 1E.

An alternative embodiment of a delivery device according to the presentinvention is shown in FIG. 2. In FIG. 2, delivery device 60 is shown ina deployed configuration at a location in vessel 70. Balloons 62 andmandrel 64 are deployed, and present in mandrel 64 are multiple deliverytubes 66 which convey the two component parts of a phase invertiblecomposition, i.e., a substrate and linker (as described below) fromseparate fluid delivery lumens in device 60 to exit ports 68. At thedistal end of each delivery tube 66 is a mixing element 67 which assistsin combining the two component parts immediately prior to exit from thedevice.

In yet another alternative embodiment, the expandable mandrel hasmultiple delivery tubes via which individual components of the two-partphase invertible composition, i.e., the substrate and linkercompositions, are delivered through separate exit ports. After deliveryof substrate material to the lesion site, the linker composition isdelivered to the site with simultaneous pulsation (i.e. inflation anddeflation) of the expandable mandrel for the purpose of mixing the twocomponents.

In yet another alternative embodiment, the substrate material isdeployed as a solid matrix on the exterior of the expandable mandrel andthen expanded to the wall of the vessel in a tubular form by expansionof the mandrel. Once in position, the linker composition is deliveredthrough the expandable mandrel and allowed to come in contact with, andmix with, the previously deployed substrate material.

The subject methods and devices of these particular representativeembodiments, as described above, find use in any application where theproduction of a biocomposite structural member in situ at a vascularsite is desired. One representative type of application in which thesubject methods find use is in the production of a biocomposite stent ata stenotic vascular site, e.g., a coronary artery, where the lesion hasbeen treated, e.g., compressed, via atherectomy or angioplastictechniques, to increase the flow of blood through the artery.

Generally the vascularized animals with which the subject invention isemployed are “mammals” or “mammalian,” where these terms are usedbroadly to describe organisms which are within the class mammalian,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), lagomorphs (e.g. rabbits) and primates(e.g., humans, chimpanzees, and monkeys). In many embodiments, theanimals or hosts, i.e., subjects (also referred to herein as patients)will be humans.

Also provided are systems for use in practicing the subject methods ofthis embodiment, where the systems at least include a fluid deliverydevice and a phase invertible fluid composition, as described above. Thesubject systems also typically include a guiding element that isemployed to position the device, e.g., in a percutaneous approachprotocol, such as a guidewire or analogous structure. Other componentsthat may be present in the subject systems include, but are not limitedto: balloon inflation means, etc.

Kits

Also provided are kits for use in practicing the subject methods, wherethe kits typically include the distinct substrate and crosslinkercomponents of a phase invertible fluid composition, as described above.The substrate and crosslinker components may be present in separatecontainers in the kit, e.g., where the substrate is present in a firstcontainer and the crosslinker is present in a second container, wherethe containers may or may not be present in a combined configuration.

The subject kits may also include a mixing device, for mixing thesubstrate and crosslinker together to produce the phase invertiblecomposition. The kits may also include a delivery device (which may ormay not include a mixing element), such as a catheter devices, asdescribed above. The kit may further include other components, e.g.,guidewires, sensor wires, etc., which may find use in practicing thesubject methods.

In addition to above-mentioned components, the subject kits typicallyfurther include instructions for using the components of the kit topractice the subject methods. The instructions for practicing thesubject methods are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In other embodiments, the instructions are present as an electronicstorage data file present on a suitable computer readable storagemedium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the Internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

The following examples are provided by way of illustration and not byway of limitation.

Experimental I. Functionality of Heat Treated Glutaraldehyde

Heat-treated glutaraldehyde was evaluated to determine crosslinkingefficiency. Glutaraldehyde solution (5% w/w) was used to crosslink asolution containing 35% albumin. The albumin was polymerized inapproximately 90 seconds, indicating the efficiency of the crosslinkingsolution of the glutaraldehyde was undisturbed.

II. Representative Uses A. Pulmonary

A rabbit was used and an experimental model for the evaluation of thematerial as a pulmonary sealant.

A sealant composition of the subject invention, consisting of albumin,collagen, oleic acid, PEI and chitosan and crosslinked withheat-processed glutaraldehyde, was prepared in accordance with themethod of invention. Concentrations for each ingredient were consistentwith the values indicated in the above examples.

The lungs of an anaesthetized rabbit were exposed and deflated.Following, a portion of the upper lobe of the lung was transected andthe cut site of the deflated lung was sealed and reinflated. The lungwas evaluated for leakage by submersion in water. Evaluation of the lungfor air leakage did not indicate any to be present, indicating theefficacy of the sealant.

B. Vascular

A rabbit was again used as an experimental model for the evaluation ofthe material as a vascular sealant.

In this experiment, the carotid arteries of an anesthetized,anticoagulated rabbit were bilaterally exposed. The artery of the leftside was punctured with a 14 F catheter. Following removal of thecatheter the hole was closed using the sealant. Alternately, the arteryof the right side was transected, and an anastomosis was created using6-0 Prolene suture. An umbilical tape was partially looped around thevessel proximal to the surgery site to momentarily reduce blood flow.

Sealant formulated to be consistent with the ranges heretofore indicatedwas applied to the puncture site using a tipped syringe. Following threeminutes, the pressure was released to expose the repair to the fullsystolic/diastolic pressure of the carotid artery. No leakage was foundto be present from the wound site.

Sealant formulated to be consistent with the ranges indicated wasapplied to the partially leaking anastomotic site of the right side ofthe experimental model. Following three minutes it was noted that theleakage stopped.

C. Cerebral Spinal Fluid

In a further experiment, a human cadaveric model was assessed foradhesion of the sealant onto the dura mata.

Following a craniotomy, the exposed dura was incised. Incision of thedura resulted in retraction of the tissue. The retracted tissue wasdrawn together, again using temporary stay sutures such that the incisededges were juxtaposed to one another. Sealant consistent with theformulations noted for this invention was prepared. The sealant wasapplied over the incision wound and the suture stays were released. Theopposing edges of the incision wound remained aligned with one another,the sealant demonstrating adequate tenacity to resist the retractiveforces of the dura. The cadaver's head was lowered placing additionalstress on the suture and the site was observed for failure of thesealant to hold the edges together. No failures were noted.

III. Tissue Compatibility Testing

A sealant composition of the subject invention, consisting of albumin,collagen, oleic acid, PEI and chitosan and crosslinked withheat-processed glutaraldehyde, was prepared in accordance with themethod of invention. Concentrations for each ingredient were consistentwith the values indicated in the above examples.

The composition was implanted in muscle tissue of a living rabbit. Themuscle tissue was then evaluated for evidence of irritation or toxicitybased on the requirements of the International Organization forStandardization 10993: Biological Evaluation of Medical Devices, Part 6:Tests for Local Effects after Implantation.

Implant samples and negative control samples were sterilized by ethyleneoxide and then degassed for 5 days. Rabbits were implanted and were theneuthanized 3 weeks later. Muscle tissues were excised and the implantsites were examined macroscopically. A microscopic evaluation ofrepresentative implant sites from each rabbit was conducted to furtherdevice any tissue response.

Under the conditions of this study, the macroscopic reaction was notsignificant as compared to the negative control implant material.Microscopically, the test article was classified as a non-irritant ascompared to the negative control article.

It is evident from the above results and discussion that the presentinvention provides an important new type of biocompatible compositionthat can be used in a variety of different applications, where benefitsof the subject compositions include, but are not limited to, lowtoxicity, high adhesion, and the like. Accordingly, the presentinvention represents a significant contribution to the art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A method comprising applying an amount of a composition comprising aheat treated aldehyde to a location of a subject.
 2. The methodaccording to claim 1, wherein the heat treated aldehyde is a heattreated dialdehyde.
 3. The method according to claim 2, wherein the heattreated dialdehyde is heat treated glutaraldehyde.
 4. The methodaccording to claim 3, wherein the composition comprises moleculesdescribed by the following formula:

wherein R₁ and R₂ are H or hydrocarbon moieties which are polymerizationproducts produced by sequential aldol reaction and dehydration of one ormore glutaraldehyde molecules.
 5. The method according to claim 1,wherein the location is a vessel.
 6. The method according to claim 1,wherein the location is an organ.
 7. The method according to claim 1,wherein the location comprises connective tissue.
 8. The methodaccording to claim 7, wherein the connective tissue comprises tissueselected from the group consisting of ligaments, tendons and bone. 9.The method according to claim 1, wherein the subject is a mammal. 10.The method according to claim 9, wherein the mammal is a human.
 11. Themethod according to claim 1, wherein the tissue is from an ungulate. 12.The method according to claim 11, wherein the ungulate is selected fromthe group consisting of cows, pigs, sheep and horses.
 13. The methodaccording to claim 1, wherein the composition comprises a proteinaceoussubstrate.
 14. The method according to claim 1, wherein the amountranges from 1 to 100 ml.
 15. A method comprising applying an amount of acomposition comprising a heat treated aldehyde to a vessel of a human.16. The method according to claim 15, wherein the heat treated aldehydeis a heat treated dialdehyde.
 17. The method according to claim 16,wherein the heat treated dialdehyde is heat treated glutaraldehyde. 18.The method according to claim 17, wherein the composition comprisesmolecules described by the following formula:

wherein R₁ and R₂ are H or hydrocarbon moieties which are polymerizationproducts produced by sequential aldol reaction and dehydration of one ormore glutaraldehyde molecules.
 19. The method according to claim 15,wherein the composition comprises a proteinaceous substrate.
 20. Themethod according to claim 15, wherein the amount ranges from 1 to 100ml.
 21. A method comprising applying an amount of a compositioncomprising a heat treated aldehyde to mammalian tissue.
 22. The methodaccording to claim 21, wherein the heat treated aldehyde is a heattreated dialdehyde.
 23. The method according to claim 22, wherein theheat treated dialdehyde is heat treated glutaraldehyde.
 24. The methodaccording to claim 23, wherein the composition comprises moleculesdescribed by the following formula:

wherein R₁ and R₂ are H or hydrocarbon moieties which are polymerizationproducts produced by sequential aldol reaction and dehydration of one ormore glutaraldehyde molecules.
 25. The method according to claim 21,wherein the composition comprises a proteinaceous substrate.
 26. Themethod according to claim 25, wherein the amount ranges from 1 to 100ml.
 27. The method according to claim 21, wherein the tissue is anorgan.
 28. The method according to claim 21, wherein the mammal is ahuman.
 29. The method according to claim 21, wherein the mammal is anungulate.
 30. The method according to claim 29, wherein the ungulate isselected from the group consisting of cows, pigs, sheep and horses.